This project, focused on characterization of the three-dimensional structures of RNA molecules in biologically relevant environments, links broadly important problems in biomedical science. First, the human immunodeficiency virus (HIV) currently infects 35 million individuals globally with roughly 3 million new infections per year. Studies of the protein components of the virus have led to the development of multiple effective drugs, but additional effective antiretroviral agents are needed. In principle, the RNA genome itself is a compelling drug target. The conserved, functional motifs in the HIV-1 genomic RNA that we have identified - and elements that we will characterize as part of this proposal - have significant promise as novel targets and pathways for antiretroviral drug development. Second, the extraordinary density of information encoded in the higher-order structure of the HIV-1 RNA genome represents a key component of the genetic code, one that we understand poorly at present. This research program will enhance our understanding of the principles that govern RNA-mediated gene regulation. It will also advance our understanding of the organization and compaction of RNA virus genomes and will illuminate principles that apply to densely organized RNAs including lncRNAs, nuclear bodies, and other regulatory elements. Third, the SHAPE probing strategy, pioneered by our lab, yields quantitative but highly specific nucleotide-resolution information, and, therefore, we are now focusing on developing multiple, alternative, complementary strategies that specifically detect true through-space and higher-order interactions in RNA. Finally, throughout this project, we will create integrated strategies that meld multiple, complementary, experimentally concise, and physically accurate structure probing technologies with user-friendly data processing to make possible facile and comprehensive functional analyses of large RNAs in relevant cellular contexts by non-expert laboratories. Our collaborative team, consisting of HIV virologists, RNA structural biologists, and experts in bioinformatics, will tackle the following Aims: (1) Analyze the structure of complete HIV-1 genomes inside authentic virions using the genome-scale SHAPE-MaP strategy; (2) identify through-space interactions across the entire HIV-1 genome by large-scale, single-molecule, correlated chemical probing experiments; (3) comprehensively define the intra- and inter-molecular interactions that mediate the RNA interactome of packaged HIV-1 genomic RNAs; and (4) create robust platform-independent software for fully automated analysis of chemical probing experiments that specifically measure through-space interactions in RNA.